Protein biomarkers for response to XPO1 inhibition in haematologic malignancies

Abstract XPO1 (Exportin‐1) is the nuclear export protein responsible for the normal shuttling of several proteins and RNA species between the nucleocytoplasmic compartment of eukaryotic cells. XPO1 recognizes the nuclear export signal (NES) of its cargo proteins to facilitate its export. Alterations of nuclear export have been shown to play a role in oncogenesis in several types of solid tumour and haematologic cancers. Over more than a decade, there has been substantial progress in targeting nuclear export in cancer using selective XPO1 inhibitors. This has resulted in recent approval for the first‐in‐class drug selinexor for use in relapsed, refractory multiple myeloma and diffuse large B‐cell lymphoma (DLBCL). Despite these successes, not all patients respond effectively to XPO1 inhibition and there has been lack of biomarkers for response to XPO1 inhibitors in the clinic. Using haematologic malignancy cell lines and samples from patients with myelodysplastic neoplasms treated with selinexor, we have identified XPO1, NF‐κB(p65), MCL‐1 and p53 protein levels as protein markers of response to XPO1 inhibitor therapy. These markers could lead to the identification of response upon XPO1 inhibition for more accurate decision‐making in the personalized treatment of cancer patients undergoing treatment with selinexor.


| INTRODUC TI ON
XPO1 is a nuclear exporter responsible for exporting proteins that contain a nuclear export signal (NES) out of the nucleus to the cytoplasm. 1-7 XPO1 contributes to normal homeostasis of eukaryotic cells by regulating the export of key proteins, 5 but alterations of XPO1 promote oncogenesis and are associated with decreased survival in cancer. [8][9][10][11] Selinexor is a selective inhibitor of XPO1 that was recently FDA-approved for multiple myeloma and diffuse large B-cell lymphoma. However, not all patients respond effectively to XPO1 inhibition, and there is a lack of biomarkers of response to XPO1 inhibitors in advanced-phase clinical trials. 12 Here, we have identified XPO1, MCL-1, NF-κB and p53 expression as potential predictive biomarkers of response to XPO1 inhibitor therapy. role in oncogenesis in several types of solid tumour and haematologic cancers. Over more than a decade, there has been substantial progress in targeting nuclear export in cancer using selective XPO1 inhibitors. This has resulted in recent approval for the first-in-class drug selinexor for use in relapsed, refractory multiple myeloma and diffuse large B-cell lymphoma (DLBCL). Despite these successes, not all patients respond effectively to XPO1 inhibition and there has been lack of biomarkers for response to XPO1 inhibitors in the clinic. Using haematologic malignancy cell lines and samples from patients with myelodysplastic neoplasms treated with selinexor, we have identified XPO1, NF-κB(p65), MCL-1 and p53 protein levels as protein markers of response to XPO1 inhibitor therapy. These markers could lead to the identification of response upon XPO1 inhibition for more accurate decision-making in the personalized treatment of cancer patients undergoing treatment with selinexor.

| Ex vivo studies in patient samples
Patients with myelodysplastic syndromes (MDS, n = 19) and oligoblastic acute myeloid leukaemia (AML with 20%-30% bone marrow blasts; n = 4) who were refractory to hypomethylating agents were treated with selinexor monotherapy on a single centre (MSKCC) IRB-approved clinical study as previously described. 11 Responses were assessed as per the modified International Working Group MDS response criteria. 13 Immunoblots were run as described above using p53 (Santa Cruz, Cat# 263), p21 (Cell Signalling, Cat#

| MDS patients are sensitive to XPO1 inhibition and exhibited similar pattern of protein profile response as seen in vitro
In this study, samples required for protein analysis were available from 13 patients with MDS. In these patients, XPO1 protein inhibition by selinexor showed differential responses across response categories, including marrow samples from complete remission (mCR; n = 6), progressive disease (PD; n = 4) and stable disease (SD; n = 3) ( Figure 2). For most responders (Figure 2A their densitometric analysis (right). Tukey's multiple comparison test was used when comparing more than two groups and T-test was used for two group comparisons (****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05).

F I G U R E 2
Western blot and densitometric analysis of serial samples from MDS patients treated with selinexor. Western blot analysis performed on serial samples from (A) patients who achieved marrow complete remission (mCR); (B) patients with progressive disease (PD) as best overall response; (C) patients who achieved stable disease (SD). Densitometric analysis of the respective WB data from mCR, PD and SD patient samples is shown below the blots. C, Cycle, D, Days, for example, in C1D1, C1 represents cycle 1 and D1 represents day 1 of treatment. EOT, end of treatment. and three of three in SD). These data are similar to the results seen in vitro, which is consistent with reports suggesting that inhibition of XPO1 modulates NF-kB signalling. There was no clear relationship between MCL-1 expression and clinical benefit (Figure 2A; Karyo-12, Karyo-08 and Karyo-21). Lastly, there was no clear correlation between p53 expression and response. In fact, overall p53 expression was low to non-detectable in most patients ( Figure 2B; Karyo-09, Karyo-03 and Karyo-22, Figure 2C; Karyo-19).

| DISCUSS ION
Previous studies have shown that XPO1, MCL-1 and NF-κB protein levels are decreased upon XPO1 inhibition 14

ACK N O WLE D G E M ENTS
JT is funded by the NCI/NIH (K08CA230319), the Doris Duke Charitable Foundation and the Edward P. Evans Foundation.

CO N FLI C T O F I NTE R E S T
JT reports speaking honorarium from Karyopharm.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data will be made available upon reasonable request to the corresponding author.